The Role Of ER Stress In The Injury Of Hypertensive Vascular Endothelial Cells

Objective In order to investigate wether the endoplasmic reticulum stress (ER Stress)was involved in endothelial cell injury under hypertension, the HUVECs（human umbiliealvein endothelial cells）were cultured. Hypertension was induced with24hours of angiotensinII (Ang II). MTT was appllied to detect the rate of apoptosis. Transmission electronmicroscope was used to observe morphological changes of endoplasmic reticulum.Immunofluorescence and western-blotting was used to detect the expression of ER Stressmaker protein PERK, IRE1α, BiP, Ero1-Lα and PDI in endothelial cells. In order to furtherconfirm the role of ER Stress in the vascular remodeling in hypertension and hypertensionendothelial cell injury, we observed the expression of PERK in aorta of different ages of ratsby immunohistochemical staining. The resluts will help to further clarify the possiblemechanism of the hypertension.Methods Part I: The HUVECs were cultured and the5-8th generation was selected.The research subgroups were as follows:①The AngⅡ1group (10-5mol/L AngⅡ+serumfree medium, culture24h)②AngⅡ2groups (10-6mol/L AngⅡ+serum free medium,culture24h)③AngⅡ3groups (10-8mol/L AngⅡ+serum free medium, culture24h)④ERStress blocker group (Add to ER Stress blocker salubrinal+serum-free medium the1h, addthe Ang Ⅱ make a final concentration of10-6mol/L for24h)⑤control group (Serum-freemedium, culture24h). Apoptosis rate was detected by MTT method. Endoplasmic reticulummorphological changes after Ang treatment was observed by Transmission electronmicroscope. Immunofluorescence and western-blotting were appllied to detect the expressionof ER Stress mark proteins, including PERK, IRE1α, BIP, Ero1-1α and PDI in endothelial cells. And we tried to observe whether the PERK pathway inhibitor salubrinal can putprotective effect on cultured HUVECs.PartⅡ：15male spontaneously hypertensive rats (SHR) and15matche Wistar Kyota rats(WKY) were used in this stduy. The arterial pressure was measured weekly once. When therats reached-8,-16and-24weeks old respectively, the thoracic aorta was removed. Thechanges of vascular endothelial cells were observed by HE staining. And the expression ofPERK, an important member of the ER stress proteins in aortic endothelial cells from ratswas observed by immunohistochemistry.Results Part I: By MTT assay, compared with the control group, Ang II treatment ofHUVECs apoptosis rate increased significantly, with the increase of Ang II concentration(10-9mol/L~10-4mol/L), HUVECs apoptosis was significantly increased (P <0.05).With thetransmission electron microscope, unclear nuclear membrane, dilation of endoplasmicreticulum, not complete particles were detected in in a large number of HUVECs aftertreatment of10-8mol/L Ang Ⅱ. With10-5mol/L AngⅡtreatment, nucler disappear, nucleolardisintegration fragmentation, organelles scarce, endoplasmic reticulum disappeare weredetected in even more HUVECs. Immunofluorescence and Western-blotting assays showedthat HUVECs normally expressed only small amounts of PERK, IRE1α, BiP and Ero1-L α,Ang Ⅱ significantly up-regulated the expression of PERK, IRE1α, BiP and Ero1-L αprotein(P<0.05), and with the AngⅡ concentration increasing, the protein expression of ERstress gradually increased in HUVECs with concentration dependent(P<0.05). α positivelycorrelation was detected in expression among PERK, IRE1α, BiP and Ero1-L (P<0.05).However, no significant differences was found in expression of PDI protein among eachobserved group (P>0.05). Out of imagine, we observed that ER stress inhibitor salubrina had noobvious protective effect on the apoptosis of HUVECs induced by AngⅡ, enen had toxicityto cultured HUVECs.PartⅡ：The blood pression was increased form8weeks of age. With the increase of age, arterial blood pressure in SHR rats increased continuously. The aortic endothelial cellsdecome rough, some of the cells loss or shedding from the vascular wall when the ratsreached16weeks and24weeks of age; By immunohistochemistry, the PERK expression inrats of SHR group were higher than that s in group WKY of the same week age,and with theincrease of age, the expression of PERK in SHR rats increased.Conclusions1. Hypertension can lead to the injury of vascular endothelial cells and increase ofapoptosis rate;2. Endoplasmic reticulum stress pathway protein PERK, IRE1α, BiP and Ero1-L may beinvolved in the vascular endothelial cell injury induced by hypertension;3. PERK, IRE1α, BiP and Ero1-L α has a synergistic effect, and together to participatein the injury of vascular endothelial induced by hypertension.